Pyrazine derivatives and uses thereof in renal monitoring

ABSTRACT

The present invention relates to pyrazine derivatives such as those represented by Formulas I and II below. 
                         
X 1  to X 4  of the compounds of Formulas I and II may be characterized as electron withdrawing groups. In contrast, Y 1  to Y 4  of the compounds of Formulas I and II may be characterized as electron donating groups. Pyrazine derivatives of the present invention may be utilized in assessing renal function. In particular, an effective amount of a pyrazine derivative of the invention may be administered into a body of a patient. The pyrazine derivative that is in the body may be exposed to visible and/or infrared light to cause spectral energy to emanate from the pyrazine derivative. This emanating spectral energy may be detected and utilized to determine renal function of the patient.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/343,231, filed on 4 Jan. 2012, which is a continuation of U.S.application Ser. No. 11/995,223, filed on 10 Jan. 2008, which is a U.S.National Stage Application under 35 U.S.C. §371 of InternationalApplication No. PCT/US07/14370, filed 20 Jun. 2007, and which claims thebenefit of priority to U.S. Provisional Application No. 60/815,712 filedon 22 Jun. 2006; and which is a continuation-in-part of U.S. patentapplication Ser. No. 11/721,186 filed on 8 Jun. 2007, which is a U.S.National Stage Application under 35 U.S.C. §371 of InternationalApplication No. PCT/US2005/046732 filed on 22 Dec. 2005, which claimspriority to U.S. Provisional Application No. 60/638,611 filed on 23 Dec.2004. Each of the above-referenced applications is expresslyincorporated by reference herein its entirety.

FIELD OF THE INVENTION

The present invention relates to pyrazine derivatives capable ofabsorbing and emanating spectral energy in the visible and/or nearinfrared spectrum. In addition, the present invention relates to methodsof using non-radioactive, exogenous agents such as the previouslymentioned pyrazine derivatives in medical procedures (e.g., themonitoring of renal function).

BACKGROUND

As a preliminary note, various publications are referenced throughoutthis disclosure by Arabic numerals in brackets. A citation correspondingto each reference number is listed following the detailed description.

Acute renal failure (ARF) is a common ailment in patients admitted togeneral medical-surgical hospitals. Approximately half of the patientswho develop ARF die, and survivors face marked increases in morbidityand prolonged hospitalization [1]. Early diagnosis is generally believedto be important, because renal failure is often asymptomatic andtypically requires careful tracking of renal function markers in theblood. Dynamic monitoring of renal functions of patients is desirable inorder to minimize the risk of acute renal failure brought about byvarious clinical, physiological and pathological conditions [2-6]. Suchdynamic monitoring tends to be particularly important in the case ofcritically ill or injured patients, because a large percentage of thesepatients tend to face risk of multiple organ failure (MOF) potentiallyresulting in death [7,8]. MOF is a sequential failure of the lungs,liver and kidneys and is incited by one or more of acute lung injury(ALI), adult respiratory distress syndrome (ARDS), hypermetabolism,hypotension, persistent inflammatory focus and sepsis syndrome. Thecommon histological features of hypotension and shock leading to MOFgenerally include tissue necrosis, vascular congestion, interstitial andcellular edema, hemorrhage and microthrombi. These changes generallyaffect the lungs, liver, kidneys, intestine, adrenal glands, brain andpancreas in descending order of frequency [9]. The transition from earlystages of trauma to clinical MOF generally corresponds with a particulardegree of liver and renal failure as well as a change in mortality riskfrom about 30% up to about 50% [10].

Traditionally, renal function of a patient has been determined usingcrude measurements of the patient's urine output and plasma creatininelevels [11-13]. These values are frequently misleading because suchvalues are affected by age, state of hydration, renal perfusion, musclemass, dietary intake, and many other clinical and anthropometricvariables. In addition, a single value obtained several hours aftersampling may be difficult to correlate with other physiologic eventssuch as blood pressure, cardiac output, state of hydration and otherspecific clinical events (e.g., hemorrhage, bacteremia, ventilatorsettings and others).

With regard to conventional renal monitoring procedures, anapproximation of a patient's glomerular filtration rate (GFR) can bemade via a 24 hour urine collection procedure that (as the namesuggests) typically requires about 24 hours for urine collection,several more hours for analysis, and a meticulous bedside collectiontechnique. Unfortunately, the undesirably late timing and significantduration of this conventional procedure can reduce the likelihood ofeffectively treating the patient and/or saving the kidney(s). As afurther drawback to this type of procedure, repeat data tends to beequally as cumbersome to obtain as the originally acquired data.

Occasionally, changes in serum creatinine of a patient must be adjustedbased on measurement values such as the patient's urinary electrolytesand osmolality as well as derived calculations such as “renal failureindex” and/or “fractional excretion of sodium.” Such adjustments ofserum creatinine undesirably tend to require contemporaneous collectionof additional samples of serum and urine and, after some delay, furthercalculations. Frequently, dosing of medication is adjusted for renalfunction and thus can be equally as inaccurate, equally delayed, and asdifficult to reassess as the measurement values and calculations uponwhich the dosing is based. Finally, clinical decisions in the criticallyill population are often equally as important in their timing as theyare in their accuracy.

It is known that hydrophilic, anionic substances are generally capableof being excreted by the kidneys [1,4]. Renal clearance typically occursvia two pathways: glomerular filtration and tubular secretion. Tubularsecretion may be characterized as an active transport process, andhence, the substances clearing via this pathway typically exhibitspecific properties with respect to size, charge and lipophilicity.

Most of the substances that pass through the kidneys are filteredthrough the glomerulus (a small intertwined group of capillaries in themalpighian body of the kidney). Examples of exogenous substances capableof clearing the kidney via glomerular filtration (hereinafter referredto as “GFR agents”) are shown in FIG. 1 and include creatinine (1),o-iodohippuran (2), and ^(99m)Tc-DTPA (3) [15-17]. Examples of exogenoussubstances that are capable of undergoing renal clearance via tubularsecretion include ^(99m)Tc-MAG3 (4) and other substances known in theart [15, 18, 19]. ^(99m)Tc-MAG3 (4) is also widely used to assess renalfunction though gamma scintigraphy as well as through renal blood flowmeasurement. As one drawback to the substances illustrated in FIG. 1,o-iodohippuran (2), ^(99m)Tc-DTPA (3) and ^(99m)Tc-MAG3 (4) includeradioisotopes to enable the same to be detected. Even if non-radioactiveanalogs (e.g., such as an analog of o-iodohippuran (2)) or othernon-radioactive substances were to be used for renal functionmonitoring, such monitoring would typically require the use ofundesirable ultraviolet radiation for excitation of those substances.

SUMMARY

In one regard, the present invention relates to transforming lipophilicfluorescent dyes into hydrophilic molecules. One concept of the presentinvention relates to molecules whose clearance properties are preferablysimilar to that of creatinine or o-iodohippuran, and to render suchmolecules hydrophilic by incorporating appropriate polar functionalitiessuch as hydroxyl, carboxyl, sulfonate, phopshonate and the like intotheir backbones. Pyrazine dyes of the invention may be characterized bysome as being desirable for renal applications because they tend to becleared from the body via the kidneys, demonstrate absorption andemission/fluorescence in the visible region, and tend to exhibitsignificant Stokes shifts. These properties allow flexibility in bothtuning a molecule to a desired wavelength and introducing a variety ofsubstituents to improve clearance properties.

In a first aspect, the present invention is directed to pyrazinederivatives of Formula I. With regard to Formula I, X¹ and X² may becharacterized as electron withdrawing substituents and may beindependently chosen from the group consisting of —CN, —CO₂R¹, —CONR²R³,—COR⁴, —NO₂, —SOR⁵, —SO₂R⁶, —SO₂OR⁷, —PO₃R⁸R⁹, —CONH(AA), and —CONH(PS).In some embodiments, at least one of X¹ and X² is either —CONH(AA) or—CONH(PS). (AA) is a polypeptide chain that includes one or more naturalor unnatural α-amino acids linked together by peptide bonds. (PS) is asulfated or non-sulfated polysaccharide chain that includes one or moremonosaccharide units connected by glycosidic linkages. Y¹ and Y² may, atleast in some embodiments, be characterized as electron donatingsubstituents and may be independently chosen from the group consistingof —OR¹⁰, —SR¹¹, —NR¹²R¹³, —N(R¹⁴)COR¹⁵, —P(R¹⁶)₃, —P(OR¹⁷)₃, andsubstituents corresponding to Formula A above. In some embodiments, atleast one of Y¹ and Y² is either —P(R¹⁶)₃ or —P(OR¹⁷)₃. Z¹ may be asingle bond, —CR¹⁸R¹⁹, —O, —NR²⁰, —NCOR²¹, —S, —SO and —SO₂. R¹ to R²¹may be any suitable substituents capable of providing and/or enhancingdesired biological and/or physicochemical properties of pyrazinederivatives of Formula I. For instance, for renal function assessment,each of the R groups of R¹ to R²¹ may independently be any one of ahydrogen atom, an anionic functional group (e.g., carboxylate,sulfonate, sulfate, phopshonate and phosphate) or a hydrophilicfunctional group (e.g., hydroxyl, carboxyl, sulfonyl, sulfonato andphosphonato). As an example, in some embodiments, R¹ to R²¹ mayindependently be selected from the group consisting of —H,—(CH₂)_(a)OR⁴³, —CH₂(CHOH)_(a)R⁴⁴, —CH₂(CHOH)_(a)CO₂H,—(CHCO₂H)_(a)CO₂H, —(CH₂)_(a)NR⁴⁵R⁴⁶, —CH[(CH₂)_(b)NH₂]_(a)CO₂H,—CH[(CH₂)_(b)NH₂]_(a)CH₂OH, —CH₂(CHNH₂)_(a)CH₂NR⁴⁷R⁴⁸,—(CH₂CH₂O)_(c)R⁴⁹, —(CH₂)_(d)CO(CH₂CH₂O)_(c)R⁵⁰, —(CH₂)_(a)SO₃H,—(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)NHSO₃H,—(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)PO₃ ⁼,—(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻ and —(CH₂)OPO₃. In such embodiments,each of R⁴³ to R⁵⁰ may independently be —H or —CH₃. ‘a’, ‘b’, ‘c’, ‘d’,‘m’ and ‘n’ may be any appropriate integers. For instance, in someembodiments, ‘a’, ‘b’, and ‘d’ may independently vary from 1 to 10, ‘c’may vary from 1 to 100, and ‘m’ and ‘n’ may independently vary from 1 to3.

A second aspect of the invention is directed to pyrazine derivatives ofFormula II. With regard to Formula II, X³ and X⁴ may be characterized aselectron withdrawing substituents and may be independently chosen fromthe group consisting of —CN, —CO₂R²², —CONR²³R²⁴, —COR²⁵, —NO₂, —SOR²⁶,—SO₂R²⁷, —SO₂OR²⁸, —PO₃R²⁹R³⁰, —CONH(AA), and —CONH(PS). In someembodiments, at least one of X³ and X⁴ is either —CONH(AA) or —CONH(PS).(AA) is a polypeptide chain that includes one or more natural orunnatural α-amino acids linked together by peptide bonds. (PS) is asulfated or non-sulfated polysaccharide chain that includes one or moremonosaccharide units connected by glycosidic linkages. Y³ and Y⁴ may, atleast in some embodiments, be characterized as electron donatingsubstituents and may be independently chosen from the group consistingof —OR³¹, —SR³², —NR³³R³⁴, —N(R³⁵)COR³⁶, —P(R³⁷)₃, —P(OR³⁸)₃, andsubstituents corresponding to Formula B above. In some embodiments, atleast one of Y³ and Y⁴ is either —P(R³⁷)₃ or —P(OR)₃. Z² may be a singlebond, —CR³⁹R⁴⁰, —O, —NR⁴¹, —NCOR⁴², —S, —SO, or —SO₂. R²² to R⁴² may beany suitable substituents capable of providing and/or enhancing desiredbiological and/or physicochemical properties of pyrazine derivatives ofFormula II. For instance, for renal function assessment, each of the Rgroups of R²² to R⁴² may independently be any one of a hydrogen atom, ananionic functional group (e.g., carboxylate, sulfonate, sulfate,phopshonate and phosphate) or a hydrophilic functional group (e.g.,hydroxyl, carboxyl, sulfonyl, sulfonato and phosphonato). As an example,in some embodiments, R²² to R⁴² may independently be selected from thegroup consisting of —H, —(CH₂)_(e)OR⁵¹, —CH₂(CHOH)_(e)R⁵²,—CH₂(CHOH)_(e)CO₂H, —(CHCO₂H)_(e)CO₂H, —(CH₂)_(e)NR⁵³R⁵⁴,—CH[(CH₂)NH₂]_(e)CO₂H, —CH[(CH₂)_(f)NH₂]_(e)CH₂OH,—CH₂(CHNH₂)_(e)CH₂NR⁵⁵R⁵⁶, —(CH₂CH₂O)_(g)R⁵⁷,—(CH₂)_(h)CO(CH₂CH₂O)_(g)R⁵⁸, —(CH₂)_(e)SO₃H, —(CH₂)_(e)SO₃ ⁻,—(CH₂)_(e)OSO₃H, —(CH₂)_(e)OSO₃ ⁻, —(CH₂)_(e)NHSO₃H, —(CH₂)_(e)NHSO₃ ⁻,—(CH₂)_(e)PO₃H₂, —(CH₂)_(e)PO₃H⁻, —(CH₂)_(e)PO₃ ⁼, —(CH₂)_(e)OPO₃H₂,—(CH₂)_(e)OPO₃H⁻ end —(CH₂)_(e)OPO₃. In such embodiments, each of R⁵ toR⁵⁸ may independently be —H or —CH₃. ‘e’, ‘f’, ‘g’, ‘h’, ‘p’ and ‘q’ maybe any appropriate integers. For instance, in some embodiments, ‘e’,‘f’, and ‘h’ may independently vary from 1 to 10, ‘g’ may vary from 1 to100, and ‘p’ and ‘q’ may independently vary from 1 to 3.

Yet a third aspect of the invention is directed to pharmaceuticallyacceptable compositions, each of which includes one or more pyrazinederivatives disclosed herein. Incidentally, the phrase “pharmaceuticallyacceptable” herein refers substances which are, within the scope ofsound medical judgment, suitable for use in contact with relevanttissues of humans and animals without undue toxicity, irritation,allergic response and the like, and are commensurate with a reasonablebenefit/risk ratio. The compositions of this third aspect may includeone or more appropriate excipients such as, but not limited to, suitablediluents, preservatives, solubilizers, emulsifiers, adjuvant and/orcarriers. One example of a composition of this third aspect may includeat least one pyrazine derivative of Formula I and at least one pyrazinederivative of Formula II. Another example of a composition of the thirdaspect may include one or more pyrazine derivatives of Formula I or oneor more pyrazine derivatives of Formula II.

Still a fourth aspect of the invention is directed to methods ofdetermining renal function using pyrazine derivatives such as thosedescribed above with regard to Formulas I and II. In these methods, aneffective amount of a pyrazine derivative is administered into the bodyof a patient (e.g., a mammal such as a human or animal subject).Incidentally, an “effective amount” herein generally refers to an amountof pyrazine derivative that is sufficient to enable renal clearance tobe analyzed. The pyrazine derivative in the body of the patient isexposed to at least one of visible and near infrared light. Due to thisexposure of the pyrazine derivative to the visible and/or infraredlight, the pyrazine derivative emanates spectral energy that may bedetected by appropriate detection equipment. This spectral energyemanating from the pyrazine derivative may be detected using anappropriate detection mechanism such as an invasive or non-invasiveoptical probe. Herein, “emanating” or the like refers to spectral energythat is emitted and/or fluoresced from a pyrazine derivative. Renalfunction can be determined based the spectral energy that is detected.For example, an initial amount of the amount of pyrazine derivativepresent in the body of a patient may be determined by amagnitude/intensity of light emanated from the pyrazine derivative thatis detected (e.g., in the bloodstream). As the pyrazine derivative iscleared from the body, the magnitude/intensity of detected lightgenerally diminishes. Accordingly, a rate at which this magnitude ofdetected light diminishes may be correlated to a renal clearance rate ofthe patient. This detection may be done periodically or in substantiallyreal time (providing a substantially continuous monitoring of renalfunction). Indeed, methods of the present invention enable renalfunction/clearance to be determined via detecting one or both a changeand a rate of change of the detected magnitude of spectral energy(indicative of an amount of the pyrazine derivative that has not beencleared) from the portion of the pyrazine derivative that remains in thebody. While this fourth aspect has been described with regard to use ofa single pyrazine derivative of the invention, it should be noted thatsome embodiments of this fourth aspect include the use of compositionsof the invention that may include one or more pyrazine derivativesdisclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates some structures of conventional renal agents.

FIG. 2 illustrates a block diagram of an assembly for assessing renalfunction.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

As mentioned above, the present invention includes pyrazine derivativesof Formula I. In a first family of embodiments, X¹ and X² areindependently selected from the group consisting of —CN, —CO₂R¹,—CONR²R³, —COR⁴, —NO₂, —SOR⁵, —SO₂R⁶, —SO₂OR⁷, —PO₃R⁸R⁹, —CONH(AA), and—CONH(PS), wherein at least one of (e.g., one of or both of) X¹ and X²is independently either —CONH(AA) or —CONH(PS). For instance, in onegroups of embodiments, at least one of X¹ and X² is —CONH(AA). Inanother group of embodiments, at least one of X¹ and X² is —CONH(PS).With further regard to this first family of embodiments, Y¹ and Y² areindependently selected from the group consisting of —OR¹⁰, —SR¹¹,—NR¹²R¹³, —N(R¹⁴)COR¹⁵, —P(R¹⁶)₃, —P(OR⁷)₃, and substituentscorresponding to Formula A above.

In some embodiments of this first family, X¹ and X² are independentlyselected from the group consisting of —CN, —CO₂R¹, —CONR²R³, —CONH(AA),and —CONH(PS), wherein at least one of X¹ and X² is independently either—CONH(AA) or —CONH(PS). Y¹ and Y² of some embodiments of the firstfamily are independently selected from the group consisting of —NR¹²R¹³and substituents corresponding to Formula A above.

In a second family of embodiments, X¹ and X² are independently selectedfrom the group consisting of —CN, —CO₂R¹, —CONR²R³, —COR⁴, —NO₂, —SOR⁵,—SO₂R⁶, —SO₂OR⁷, —PO₃R⁸R⁹, —CONH(AA), and —CONH(PS). Further, Y¹ and Y²are independently selected from the group consisting of —OR¹⁰, —SR¹¹,—NR¹²R¹³, —N(R¹⁴)COR¹⁵, —P(R¹⁶)₃, —P(OR¹⁷)₃, and substituentscorresponding to Formula A above, wherein at least one of (e.g., one ofor both of) Y¹ and Y² is independently either —P(R¹⁶)₃ or —P(OR¹⁷)₃. Forinstance, in one group of embodiments, at least one of Y¹ and Y² is—P(R¹⁶)₃. In another group of embodiments, at least one of Y¹ and Y² is—P(OR¹⁷)₃.

In some embodiments of this second family, X¹ and X² are independentlyselected from the group consisting of —CN, —CO₂R¹, —CONR²R³; —CONH(AA),and —CONH(PS). In some embodiments of the second family, one of Y¹ andY² is —P(R¹⁶)₃ or —P(OR¹⁷)₃, and the other of Y¹ and Y² is —NR¹²R¹³ or asubstituent corresponding to Formula A above.

With regard to the above-described first and second families, Z¹ isselected from the group consisting of a single bond, —CR¹⁸R¹⁹, —O,—NR²⁰, —NCOR²¹, —S, —SO and —SO₂. In some embodiments, Z¹ is selectedfrom the group consisting of —O, —NR²⁰, —S, —SO, and —SO₂. In otherembodiments, Z¹ is selected from the group consisting of —O and —NR²⁰.

R¹ to R²¹ of the first and second families are independently selectedfrom the group consisting of —H, —(CH₂)OR⁴³, —CH₂(CHOH)_(a)R⁴⁴,—CH₂(CHOH)_(a)CO₂H, —(CHCO₂H)_(a)CO₂H, —(CH₂)_(a)NR⁴⁵R⁴⁶,—CH[(CH₂)_(b)NH₂]_(a)CO₂H, —CH[(CH₂)_(b)NH₂]_(a)CH₂OH,—CH₂(CHNH₂)_(a)CH₂NR⁴⁷R⁴⁸, —(CH₂CH₂O)_(c)R⁴⁹, —(CH₂)CO(CH₂CH₂O)_(c)R⁵⁰,—(CH₂)_(a)SO₃H, —(CH₂)SO₃ ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)OSO₃ ⁻,—(CH₂)_(a)NHSO₃H, —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻,—(CH₂)_(a)PO₃ ⁼, —(CH₂)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻ and —(CH₂)_(a)OPO₃. Insuch embodiments, R⁴³ to R⁵⁰ are independently —H or —CH₃. In one groupof embodiments, R¹ to R²¹ of the first and second families areindependently selected from the group consisting of —H, —(CH₂)_(a)OR⁴³,—CH₂(CHOH)_(a)R⁴⁴, —CH₂(CHOH)_(a)CO₂H, —(CHCO₂H)_(a)CO₂H,—(CH₂)_(a)NR⁴⁵R⁴⁶, —CH[(CH₂)_(b)NH₂]_(a)CO₂H,—CH[(CH₂)_(b)NH₂]_(a)CH₂OH, —CH₂(CHNH₂)_(a)CH₂NR⁴⁷R⁴⁸,—(CH₂CH₂O)_(c)R⁴⁹, —(CH₂)_(d)CO(CH₂CH₂O)_(c)R⁵⁰. In another group ofembodiments, R¹ to R²¹ are independently selected from the groupconsisting of —H, —(CH₂)_(a)OR⁴³, —CH₂(CHOH)_(a)R⁴⁴, —(CH₂)_(a)NR⁴⁵R⁴⁶,—(CH₂CH₂O)_(c)R⁴⁹, and —(CH₂)_(d)CO(CH₂CH₂O)_(d)R⁵⁰. In still anothergroup of embodiments, R¹ to R²¹ are independently selected from thegroup consisting of —H, —(CH₂)_(a)OR⁴³, —CH₂(CHOH)_(aR) ⁴⁴,—(CH₂)_(a)NR⁴⁵R⁴⁶, and —(CH₂)_(d)CO(CH₂CH₂O)_(d)R⁵⁰.

Still with regard to these first and second families, ‘a’, ‘b’, and ‘d’independently vary from 1 to 10, ‘c’ varies from 1 to 100, and ‘m’ and‘n’ independently varies from 1 to 3. In some embodiments, each of ‘a’,‘b’, and ‘d’ independently varies from 1 to 6. In some embodiments, ‘c’varies from 1 to 20. In some embodiments, ‘m’ and ‘n’ are independently0 or 1.

(AA) is polypeptide chain including one or more natural or unnaturalα-amino acids linked together by peptide bonds. The polypeptide chain(AA) may be a homopolypeptide chain or a heteropolypeptide chain, andmay be any appropriate length. For instance, in some embodiments, thepolypeptide chain may include 1 to 100α-amino acid(s), 1 to 90α-aminoacid(s), 1 to 80α-amino acid(s), 1 to 70α-amino acid(s), 1 to 60α-aminoacid(s), 1 to 50α-amino acid(s), 1 to 40α-amino acid(s), 1 to 30α-aminoacid(s), 1 to 20α-amino acid(s), or even 1 to 10α-amino acid(s). In someembodiments, the α-amino acids of the polypeptide chain (AA) areselected from the group consisting of aspartic acid, asparigine,arginine, histidine, lysine, glutamic acid, glutamine, serine, andhomoserine. In some embodiments, the α-amino acids of the polypeptidechain (AA) are selected from the group consisting of aspartic acid,glutamic acid, serine, and homoserine. In some embodiments, thepolypeptide chain (AA) refers to a single amino (e.g., either asparticacid or serine).

(PS) is a sulfated or non-sulfated polysaccharide chain including one ormore monosaccharide units connected by glycosidic linkages. Thepolysaccharide chain (PS) may be any appropriate length. For instance,in some embodiments, the polysaccharide chain may include 1 to 100monosaccharide unit(s), 1 to 90 monosaccharide unit(s), 1 to 80monosaccharide unit(s), 1 to 70 monosaccharide unit(s), 1 to 60monosaccharide unit(s), 1 to 50 monosaccharide unit(s), 1 to 40monosaccharide unit(s), 1 to 30 monosaccharide unit(s), 1 to 20monosaccharide unit(s), or even 1 to 10 monosaccharide unit(s). In someembodiments, the polysaccharide chain (PS) is a homopolysaccharide chainconsisting of either pentose or hexose monosaccharide units. In otherembodiments, the polysaccharide chain (PS) is a heteropolysaccharidechain consisting of one or both pentose and hexose monosaccharide units.In some embodiments, the monosaccharide units of the polysaccharidechain (PS) are selected from the group consisting of glucose, fructose,mannose, xylose and ribose. In some embodiments, the polysaccharidechain (PS) refers to a single monosaccharide unit (e.g., either glucoseor fructose).

The present invention is also includes pyrazine derivativescorresponding to Formula II above. In a third family of embodiments ofthe invention, X³ and X⁴ are independently selected from the groupconsisting of —CN, —CO₂R²², —CONR²³R²⁴, —COR²⁵, —NO₂, —SOR²⁶, —SO₂R²⁷,—SO₂OR²⁸, —PO₃R²⁹R³⁰, —CONH(AA), and —CONH(PS), wherein at least one of(e.g., one of or both of) X³ and X⁴ is independently either —CONH(AA) or—CONH(PS). For instance, in one groups of embodiments, at least one ofX³ and X⁴ is —CONH(AA). In another group of embodiments, at least one ofX³ and X⁴ is —CONH(PS). With further regard to this first family ofembodiments, Y³ and Y⁴ are independently selected from the groupconsisting of —OR³¹, —SR³², —NR³³R³⁴, —N(R³⁵)COR³⁶, —P(R³⁷)₃, —P(OR³⁸)₃,and substituents corresponding to Formula B above.

In some embodiments of this third family, X³ and X⁴ are independentlyselected from the group consisting of —CN, —CO₂R²², —CONR²³R²⁴,—CONH(AA)_(r), and —CONH(PS)_(s), wherein at least one of X³ and X⁴ isindependently either —CONH(AA) or —CONH(PS). Y³ and Y⁴ of someembodiments of the third family are independently selected from thegroup consisting of —NR³¹R³² and substituents corresponding to Formula Babove.

In still a fourth family of embodiments, X³ and X⁴ are independentlyselected from the group consisting of —CN, —C₂R²², —CONR²³R²⁴, —COR²⁵,—NO₂, —SOR²⁶, —SO₂R²⁷, —SO₂OR²⁸, —PO₃R²⁹R³⁰, —CONH(AA), and —CONH(PS).Further, Y³ and Y⁴ are independently selected from the group consistingof —OR³¹, —SR³², —NR³³R³⁴, —N(R³⁵)COR³⁶, —P(R³⁷)₃, —P(OR³⁸)₃, andsubstituents corresponding to Formula B above, wherein at least one ofY³ and Y⁴ is independently either —P(R³⁷)₃ or —P(OR³⁸)₃. For instance,in one group of embodiments, at least one of Y³ and Y⁴ is —P(R³⁷)₃. Inanother group of embodiments, at least one of Y³ and Y⁴ is —P(OR³⁸)₃.

In some embodiments of this fourth family, X³ and X⁴ are independentlyselected from the group consisting of —CN, —CO₂R²², —CONR²³R²⁴,—CONH(AA), and —CONH(PS). In some embodiments of the fourth family, oneof Y³ and Y⁴ is —P(R³⁷)₃ or —P(OR³⁸)₃, and the other of Y³ and Y⁴ is—NR³¹R³² or a substituent corresponding to Formula B above.

With regard to the above-described third and fourth families, Z² isselected from the group consisting of a single bond, —CR³⁹R⁴⁰, —O,—NR⁴¹, —NCOR⁴², —S, —SO, and —SO₂. In some embodiments, Z² is selectedfrom the group consisting of —O, —NR⁴¹, —S, —SO and —SO₂. In someembodiments, Z² is selected from the group consisting of —O and —NR⁴¹.

R²² to R⁴² are independently selected from the group consisting of —H,—(CH₂)_(e)OR⁵¹, —CH₂(CHOH)_(e)R⁵², —CH₂(CHOH)_(e)CO₂H,—(CHCO₂H)_(e)CO₂H, —(CH₂)_(e)NR⁵³R⁵⁴, —CH[(CH₂)_(f)NH₂]_(e)CO₂H,—CH[(CH₂)_(r)NH₂]_(e)CH₂OH, —CH₂(CHNH₂)_(e)CH₂NR⁵⁵R⁵⁶, —(CH₂H₂O)_(g)R⁵⁷,—(CH₂)_(h)CO(CH₂CH₂O)_(g)R⁵⁸, —(CH₂)_(e)SO₃H, —(CH₂)_(e)SO₃ ⁻,—(CH₂)_(e)OSO₃H, —(CH₂)_(e)OSO₃ ⁻, —(CH₂)_(e)NHSO₃H, —(CH₂)_(e)NHSO—,—(CH₂)_(e)PO₃H₂, —(CH₂)_(e)PO₃H⁻, —(CH₂)_(e)PO₃ ⁼, —(CH₂)_(e)OPO₃H₂,—(CH₂)_(e)OPO₃H⁻ end —(CH₂)_(e)OPO₃. In such embodiments, R⁵¹ to R⁵⁸ areindependently —H or —CH₃. In one group of embodiments, R²² to R⁴² areindependently selected from the group consisting of —H, —(CH₂)_(e)OR⁵¹,—CH₂(CHOH)_(e)R⁵², —CH₂(CHOH)_(e)CO₂H, —(CHCO₂H)_(e)CO₂H,—(CH₂)_(e)NR⁵³R⁵⁴, —CH[(CH₂)NH₂]_(e)CO₂H, —CH[(CH₂)_(f)NH₂]_(e)CH₂OH,—CH₂(CHNH₂)_(e)CH₂NR⁵⁵R⁵⁶, —(CH₂CH₂O)_(g)R⁵⁷, and—(CH₂)_(h)CO(CH₂CH₂O)_(g)R⁵⁸. In another group of embodiments, R²² toR⁴² are independently selected from the group consisting of —H,—(CH₂)_(e)OR⁵¹, —CH₂(CHOH)_(e)R⁵², —(CH₂)_(e)NR⁵³R⁵⁴, —(CH₂CH₂O)_(g)R⁵⁷,and —(CH₂)_(h)CO(CH₂CH₂O)_(g)R⁵⁸. In still another group of embodiments,R²² to R⁴² are independently selected from the group consisting of —H,—(CH₂)_(e)OR⁵¹, —CH₂(CHOH)_(e)R⁵², —(CH₂)_(e)NR⁵³R⁵⁴, and—(CH₂)_(h)CO(CH₂CH₂O)_(g)R⁵⁸.

Still with regard to the third and fourth families, ‘e’, ‘f’, and ‘h’independently vary from 1 to 10, ‘g’ varies from 1 to 100, and ‘p’ and‘q’ independently vary from 1 to 3. In some embodiments, ‘e’, ‘f’, and‘h’ independently vary from 1 to 6. In some embodiments, ‘g’ varies from1 to 20. In some embodiments, ‘m’ and ‘n’ are independently 0 or 1.

As with the first and second families of embodiments described above,(AA) of the third and fourth families is a polypeptide chain includingone or more natural or unnatural α-amino acids linked together bypeptide bonds. Accordingly, the description of (AA) with reference tothe first and second families of embodiments above applies to (AA) ofthe third and fourth families of embodiments as well. Likewise, (PS) ofthe third and fourth families is a sulfated or non-sulfatedpolysaccharide chain including one or more monosaccharide unitsconnected by glycosidic linkages. As such, the description of (PS) withreference to the first and second families of embodiments above appliesto (PS) of the third and fourth families of embodiments as well.

Syntheses of pyrazine derivatives, in general, have been previouslystudied [27] and described [25, 26, 28, 29]. Preparation procedures forsome of the pyrazine derivatives of the present invention, usingprocedures similar to the above references, are described later inExamples 1 to 11. It is noteworthy that the alkylation of the electrondonating amino group in cyano- or carboxypyrazines has a profound effecton electronic transition of the pyrazine chromophore in that thedialkylation of the amino group in 2,5-diamino-3,5-dicyanopyrazineproduces large bathochromic shift of about 40-60 nm. It is alsonoteworthy that the pyrrolidino and piperidio derivatives exhibitsubstantial difference in their UV spectra in that the former exhibits abathochromic shift of about 34 nm. These results could be explained onthe basis that the highest occupied molecular orbital (HOMO) of thealkylated aminopyrazine is destabilized compared to the parent aminocompound. Therefore, based on the above premise, it is predicted thatpyrazine derivatives containing highly strained azacycloalkylsubstituents, which were not disclosed previously, exhibit largerbathochromic shifts compared to unstrained cyclic analogs.

In accordance with the present invention, one protocol for assessingphysiological function of body cells includes administering an effectiveamount of a pyrazine derivative represented by Formula I or II into abody of a patient. An appropriate dosage of the pyrazine derivate thatis administered to the patient is readily determinable by one ofordinary skill in the art and may vary according to the clinicalprocedure contemplated, generally ranging from about 1 nanomolar toabout 100 micromolar. The administration of the pyrazine derivative tothe patient may occur in any of a number of appropriate fashionsincluding, but not limited to: (1) intravenous, intraperitoneal, orsubcutaneous injection or infusion; (2) oral administration; (3)transdermal absorption through the skin; and (4) inhalation.

Pyrazine derivatives of this invention can be administered as solutionsin most pharmaceutically acceptable intravenous vehicles known in theart. Pharmaceutically acceptable vehicles that are well known to thoseskilled in the art include, but are not limited to, 0.01-0.1M phosphatebuffer or 0.8% saline. Additionally, pharmaceutically acceptablecarriers may be aqueous or non-aqueous solutions, suspensions,emulsions, or appropriate combinations thereof. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Examples of aqueous carriers are water, alcoholic/aqueous solutions,emulsions or suspensions, including saline and buffered media. Exemplaryparenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils. Exemplaryintravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

Suitable diluents, preservatives, solubilizers, emulsifiers, adjuvantand/or carriers are also suitable excipients. Such compositions areliquids or lyophilized or otherwise dried formulations and includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength, additives such as albumin or gelatin to preventabsorption to surfaces, detergents (e.g., Tween 20, Tween 80, PluronicF68, bile acid salts), solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulkingsubstances or tonicity modifiers (e.g., lactose, mannitol), complexationwith metal ions, or incorporation of the material into or ontoparticulate preparations of polymeric compounds such as polylactic acid,polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions may likely influence the physical state,solubility, stability, rate of in vivo release, and/or rate of in vivoclearance.

Still referring to the above-mentioned protocol, the pyrazine derivativeis exposed to visible and/or near infrared light. This exposure of thepyrazine derivate to light may occur at any appropriate time butpreferably occurs while the pyrazine derivative is located in the body.Due to this exposure of the pyrazine derivate to the visible and/orinfrared light, the pyrazine derivate emanates spectral energy (e.g.,visible and/or near infrared light) that may be detected by appropriatedetection equipment. The spectral energy emanated from the pyrazinederivative tends to exhibit a wavelength range greater than a wavelengthrange absorbed by the pyrazine derivative. For example, if an embodimentof the pyrazine derivative absorbs light of about 700 nm, the pyrazinederivative may emit light of about 745 nm.

Detection of the pyrazine derivate (or more particularly, the lightemanating therefrom) may be achieved through optical fluorescence,absorbance or light scattering procedures known in the art. In oneembodiment, this detection of the emanated spectral energy may becharacterized as a collection of the emanated spectral energy and ageneration of electrical signal indicative of the collected spectralenergy. The mechanism(s) utilized to detect the spectral energy from thepyrazine derivative that is present in the body may be designed todetect only selected wavelengths (or wavelength ranges) and/or mayinclude one or more appropriate spectral filters. Various catheters,endoscopes, ear clips, hand bands, head bands, surface coils, fingerprobes and the like may be utilized to expose the pyrazine derivative tolight and/or to detect the light emanating therefrom [30]. Thisdetection of spectral energy may be accomplished at one or more timesintermittently or may be substantially continuous.

Renal function of the patient can be determined based on the detectedspectral energy. This can be achieved by using data indicative of thedetected spectral energy and generating an intensity/time profileindicative of a clearance of the pyrazine derivative from the body. Thisprofile may be correlated to a physiological or pathological condition.For example, the patient's clearance profiles and/or clearance rates maybe compared to known clearance profiles and/or rates to assess thepatient's renal function and to diagnose the patient's physiologicalcondition. In the case of analyzing the presence of the pyrazinederivative in bodily fluids, concentration/time curves may be generatedand analyzed (preferably in real time) using an appropriatemicroprocessor to diagnose renal function.

Physiological function can be assessed by: (1) comparing differences inmanners in which normal and impaired cells remove a pyrazine derivativeof the invention from the bloodstream; (2) measuring a rate or anaccumulation of a pyrazine derivative of the invention in the organs ortissues; and/or (3) obtaining tomographic images of organs or tissueshaving a pyrazine derivative of the invention associated therewith. Forexample, blood pool clearance may be measured non-invasively fromconvenient surface capillaries such as those found in an ear lobe or afinger or can be measured invasively using an appropriate instrumentsuch as an endovascular catheter. Accumulation of a pyrazine derivativeof the invention within cells of interest can be assessed in a similarfashion.

A modified pulmonary artery catheter may also be utilized to, interalia, make the desired measurements [32] of spectral energy emanatingfrom a pyrazine derivative of the invention. The ability for a pulmonarycatheter to detect spectral energy emanating from a pyrazine derivativeof the invention is a distinct improvement over current pulmonary arterycatheters that measure only intravascular pressures, cardiac output andother derived measures of blood flow. Traditionally, critically illpatients have been managed using only the above-listed parameters, andtheir treatment has tended to be dependent upon intermittent bloodsampling and testing for assessment of renal function. These traditionalparameters provide for discontinuous data and are frequently misleadingin many patient populations.

Modification of a standard pulmonary artery catheter only requiresmaking a fiber optic sensor thereof wavelength-specific. Catheters thatincorporate fiber optic technology for measuring mixed venous oxygensaturation exist currently. In one characterization, it may be said thatthe modified pulmonary artery catheter incorporates awavelength-specific optical sensor into a tip of a standard pulmonaryartery catheter. This wavelength-specific optical sensor can be utilizedto monitor renal function-specific elimination of a designed opticallydetectable chemical entity such as the pyrazine derivatives of thepresent invention. Thus, by a method analogous to a dye dilution curve,real-time renal function can be monitored by the disappearance/clearanceof an optically detected compound.

The following examples illustrate specific embodiments of the invention.As would be apparent to skilled artisans, various changes andmodifications are possible and are contemplated within the scope of theinvention described.

Example 1 Preparation of3,6-diamino-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide

A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (200 mg, 1.01mmol), bis-2-(methoxyethyl)amine (372 μL, 335.5 mg, 2.52 mmol), HOBt-H₂O(459 mg, 3.00 mmol), and EDC-HCl (575 mg, 3.00 mmol) were stirredtogether in DMF (20 mL) for 1 h at room temperature. The mixture wasconcentrated to dryness and the residue was partitioned with EtOAc andwater. The layers were separated and the EtOAc solution was washed withsaturated NaHCO₃ and brine. The solution was dried over anhydrousNa₂SO₄, filtered and concentrated. Purification by radial flashchromatography (SiO₂, 10/1 CHCl₃-MeOH) afforded 228.7 mg (53% yield) ofExample 1 as an orange foam: ¹H NMR (300 MHz, CDCl₃), δ 4.92 (s, 4H),3.76 (apparent t, J=5.4 Hz, 4H), 3.70 (apparent t, J=5.6 Hz, 4H), 3.64(apparent t, J=5.4 Hz, 4H), 3.565 (apparent t, J=5.4 Hz), 3.67 (s, 6H),3.28 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 167.6 (s), 145.6 (s), 131.0 (s),72.0 (t), 70.8 (t), 59.2 (q), 49.7 (t), 47.1 (t). LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=3.14min on 30 mm column, (M+H)⁺=429. UV/vis (100 μM in PBS) λ_(abs)=394 nm.Fluorescence (100 nm) λ_(ex)=394 nm λ_(em)=550 nm.

Example 23,6-diamino-N²,N⁵-bis(2,3-dihydroxypropyl)pyrazine-2,5-dicarboxamide

Step 1 Synthesis of3,6-diamino-N²,N⁵-bis((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)pyrazine-2,5-dicarboxamide

A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (350 mg, 1.77mmol), racemic (2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (933 μL, 944mg, 7.20 mmol), HOBt-H₂O (812 mg, 5.3 mmol), and EDC-HCl (1.02 g, 5.32mmol) were stirred together in DMF (20 mL) for 16 h at room temperature.The mixture was concentrated to dryness and the residue was partitionedwith EtOAc and water. The layers were separated and the EtOAc solutionwas washed with saturated NaHCO₃ and brine. The solution was dried overanhydrous Na₂SO₄, filtered and concentrated to afford 665 mg (88% yield)of the bis-amide diastereomeric pair as a yellow solid: ¹NMR (300 MHz,CDC3) δ 8.38 (t, J=5.8 Hz, 2H), 6.55 (s, 4H), 4.21 (quintet, J=5.8 Hz,2H), 3.98 (dd, J=8.4 Hz, 6.3 Hz, 2H), 3.65 (dd, J=8.4 Hz, J=5.8 Hz, 2H),3.39 (apparent quartet—diastereotopic mixture, J=5.9 Hz, 4H), 1.35 (s,6H), 1.26 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 165.7 (s), 146.8 (s), 126.8(s), 109.2 (s), 74.8 (d), 67.2 (t), 42.2, 41.1 (t—diastereotopic pair),27.6 (q), 26.1 (q).

Step 2

The product from Step 1 was dissolved in THF (100 mL) and treated with1.0N HCl (2 mL). After hydrolysis was complete, the mixture was treatedwith K₂CO₃ (1 g) and stirred for 1 h and filtered through a plug of C18with using methanol. The filtrate was concentrated to dryness and theresidue was triturated with MeOH (50 mL). The solids were filtered anddiscarded and the residue was treated with ether (50 mL). Theprecipitate was collected by filtration and dried at high vacuum. Thismaterial was purified by radial flash chromatography to afford 221 mg(36% yield) of Example 2 as a orange solid: ¹NMR (300 MHz, DMSO-d₆) δ8.00 (bm, 6H), 5.39 (bs, 2H), 4.88 (bs, 2H), 3.63-3.71 (complex m, 2H),3.40 (dd, J=11.1, 5.10 Hz, 2H), 3.28 (dd, J=11.1, 6.60 Hz, 2H), 2.92(dd, J=12.6, 3.3 Hz, 2H), 2.65 (dd, J=12.6, 8.4 Hz, 2H). LCMS (5-95%gradient acetonitrile in 0.1% TFA over 10 min), single peak retentiontime=4.13 min on 30 mm column, (M+H)⁺=345. UV/vis (100 μM in H₂O)λ_(abs)=432 nm. Fluorescence λ_(ex)=432 nm, λ_(em)=558 nm.

Example 3 3,6-Diamino-N²,N⁵-bis(serine)-pyrazine-2,5-dicarboxamide

Step 1 Synthesis of 3,6-Diamino-N²,N⁵-bis(O-benzylserine methylester)-pyrazine-2,5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (300 mg, 1.24mmol), Ser(OBn)-OMe-HCl salt (647 mg, 2.64 mmol), HOBt-H₂O (570 mg, 3.72mmol) and EDC-HCl (690 mg, 3.60 mmol) in DMF (25 mL) was treated withTEA (2 mL). The resulting mixture was stirred for 16 h and concentrated.Work up as in Example 1 afforded 370 mg (51% yield) of the bisamide as abright yellow powder: ¹H NMR (300 MHz, CDCl₃) δ 8.47 (d, J=8.74 Hz, 2H),7.25-7.37 (complex m, 10H), 5.98 (bs, 4H), 4.85 (dt, J=8.7, 3.3 Hz, 2H),4.56 (ABq, J=12.6, Hz, Δν=11.9 Hz, 4H), 3.99 (one half of an ABq of d,J=8.7, 3.3, Δν obscured, 2H), 3.76-3.80 (one half of an ABq—obscured,2H), 3.78 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) □ 170.5 (s), 165.1 (s), 146.8(s), 138.7 (s) 128.6 (d), 128.1 (d), 127.8 (d), 126.9 (s), 73.5 (t),69.8 (t), 53.0 (q), 52.9 (q). LCMS (5-95% gradient acetonitrile in 0.1%TFA over 10 min), single peak retention time=4.93 min on 30 mm column,(M+H)⁺=581.

Step 2 Synthesis of3,6-Diamino-N²,N⁵-bis(O-benzylserine)-pyrazine-2,5-dicarboxamide

The product from step 1 (370 mg, 0.64 mmol) in THF (10 mL) was treatedwith 1.0N sodium hydroxide (2.5 mL). After stirring at room temperaturefor 30 min, the reaction was judged complete by TLC. The pH was adjustedto approximately 2 by the addition of 1.0N HCl and the resultingsolution was extracted (3×) with EtOAc. The layers were combined, driedover sodium sulfate, filtered and concentrated to afford 353 mg (100%yield) of the diacid as an orange foam: LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), retention time=4.41 min on 30 mmcolumn, (M+H)⁴=553.

Step 3

To the product from step 2 (353 mg, 0.64 mmol) in methanol (20 mL) wasadded 5% Pd/C (300 mg) and ammonium formate (600 mg). The resultingreaction was heated at reflux for 2 h. The reaction was cooled to roomtemperature, filtered through a plug of celite and concentrated. Theresidue was recrystallized from methanol-ether to provide 191 mg (80%yield) of Example 3 as a yellow foam: ¹NMR (300 MHz, DMSO-d₆) δ 8.48 (d,J=6.9 Hz, 2H), 6.72 (bs, 4H), 3.95 (apparent quartet, J=5.1 Hz, 2H),3.60 (apparent ABq of doublets; down-field group centered at 3.71,J=9.9, 5.1 Hz, 2H; up-field group centered at 3.48, J=9.9, 6.3 Hz, 2H).¹³C NMR (75 MHz, CDCl3) δ 172.9 (s), 164.9 (s), 147.0 (s), 127.0 (s),62.9 (d), 55.7 (t). LCMS (5-95% gradient acetonitrile in 0.1% TFA over10 min), single peak retention time=1.45 min on 30 mm column,(M+H)⁺=373. UV/vis (100 μM in PBS) λ_(abs)=434 nm. Fluorescenceλ_(ex)=449 nm, λ_(em)=559 nm.

Example 43,6-bis(bis(2-methoxyethyl)amino)-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide bis TFA salt

Step 1 Synthesis of 3,6-dibromopyrazine-2,5-dicarboxylic acid

3,6-Diaminopyrazine-2,5-dicarboxylic acid (499 mg, 2.52 mmol) wasdissolved in 48% hydrobromic acid (10 mL) and cooled to 0° C. in anice-salt bath. To this stirred mixture was added a solution of sodiumnitrite (695 mg, 10.1 mmol) in water (10 mL) dropwise so that thetemperature remains below 5° C. The resulting mixture was stirred for 3h at 5-15° C., during which time the red mixture became a yellowsolution. The yellow solution was poured into a solution of cupricbromide (2.23 g, 10.1 mmol) in water (100 mL) and the resulting mixturewas stirred at room temperature. After an addition 3 h, the aqueousmixture was extracted with EtOAc (3×). The combined extracts were dried(Na₂SO₄), filtered and concentrated to afford 440 mg (54% yield)3,6-dibromopyrazine-2,5-dicarboxylic acid as a pale yellow solid: ¹³CNMR (75 MHz, CDCl₃) δ 164.3 (s), 148.8 (s), 134.9 (s). HPLC (5-95%gradient acetonitrile in 0.1% TFA over 10 min), single peak retentiontime=2.95 min on 250 mm column.

Step 2 Synthesis of3-(Bis(2-methoxyethyl)amino)-6-bromo-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide

The product from step 1 (440 mg, 1.36 mmol) was dissolved in DMF (25mL), treated with HOBt-H₂O (624 mg, 4.08 mmol), and EDC-HCl (786 mg,4.10 mmol) and stirred for 30 min at room temperature.Bis(2-methoxylethyl)amine (620 mL, 559 mg, 4.20 mmol) was added and theresulting mixture was stirred at room temperature for 16 h andconcentrated. The residue was partitioned with water and EtOAc. TheEtOAc layer was separated and the aqueous was extracted again withEtOAc. The combined organic layers were washed with 0.5N HCl, saturatedsodium bicarbonate, and brine. The organic layer was dried (Na₂SO₄),filtered and concentrated to afford 214 mg of3-(bis(2-methoxyethyl)amino)-6-bromo-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide(26% yield) as a brown oil: LCMS (5-95% gradient acetonitrile in 0.1%TFA over 10 min), single peak retention time=3.85 min on 30 mm column,(M+H)⁺=608.

Step 3

To the product from step 2 (116 mg, 0.19 mmol) was addedbis(2-methoxylethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and a “spatulatip” of Pd(PPh₃)₄. The resulting mixture was heated to 140° C. for 2 h.The reaction was cooled and concentrated. The residue was purified byflash chromatography (SiO₂, 10/1 CHCl₃-MeOH). The resulting material waspurified again by reverse phase medium pressure chromatography (C18,10-50% manual gradient acetonitrile in 0.1% TFA) to afford 12 mg (10%yield) of Example 4 as an orange-brown film: LCMS (15-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=3.85min on 250 mm column, (M+H)⁺=661. UV/vis (100 kM in PBS) λ_(abs)=434 nm.Fluorescence λ_(ex)=449 nm, λ_(em)=559 nm.

Example 5 3,6-diamino-N²,N⁵-bis(2-aminoethyl)pyrazine-2,5-dicarboxamidebis TFA salt

Step 1 Synthesis of3,6-diamino-N²,N⁵-bis[2-(tert-butoxycarbonyl)aminoethyl]pyrazine-2,5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (500 mg, 2.07mmol), tert-butyl 2-aminoethylcarbamate (673 mg, 4.20 mmol), HOBt-H₂O(836 mg, 5.46 mmol) and EDC-HCl (1.05 g, 5.48 mmol) in DMF (25 mL) wasstirred for 16 h and concentrated. Work up as in Example 1 afforded 770mg (76% yield) of the bisamide as an orange foam: ¹NMR (300 MHz,DMSO-d₆) major comformer, δ 8.44 (t, J=5.7 Hz, 2H), 6.90 (t, J=5.7 Hz,2H), 6.48 (bs, 4H), 2.93-3.16 (complex m, 8H), 1.37 (s, 9H), 1.36 (s,9H). ¹³C NMR (75 MHz, DMSO-d₆), conformational isomers δ 165.1 (s),155.5 (bs), 155.4 (bs), 146.0 (s), 126.2 (s), 77.7 (bs), 77.5 (bs), 45.2(bt), 44.5 (bt), 28.2 (q).

Step 2

To the product from step 1 (770 mg, 1.60 mmol) in methylene chloride(100 mL) was added TFA (25 mL) and the reaction was stirred at roomtemperature for 2 h. The mixture was concentrated and the residue takenup into methanol (15 mL). Ether (200 mL) was added and the orange solidprecipitate was isolated by filtration and dried at high vacuum toafford 627 mg (77% yield) of Example 5 as an orange powder: ¹NMR (300MHz, DMSO-d₆) δ 8.70 (t, J=6 Hz, 2H), 7.86 (bs, 6H), 6.50 (bs, 4H),3.46-3.58 (m, 4H), 3.26-3.40 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 166.4(s), 146.8 (s), 127.0 (s), 39.4 (t), 37.4 (t). LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=3.62min on 30 mm column, (M+H)⁺=283. UV/vis (100 μM in PBS) λ_(abs)=435 nm.Fluorescence (100 nM) λ_(ex)=449 nm, λ_(em)=562 nm.

Example 6 3,6-Diamino-N²,N⁵-bis(D-Aspartate)-pyrazine-2,5-dicarboxamide

Step 1 Synthesis of 3,6-Diamino-N²,N⁵-bis(benzylD-Obenzyl-Aspartate)-pyrazine-2,5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (600 mg, 2.48mmol), Asp(OBn)-OMe-p-TosH salt (2.43 g, 5.00 mmol), HOBt-H₂O (919 mg,6.00 mmol) and EDC-HCl (1.14 g, 5.95 mmol) in DMF (50 mL) was treatedwith TEA (4 mL). The resulting mixture was stirred over night at roomtemperature. The reaction mixture was concentrated and the residue waspartitioned with water and EtOAc. The EtOAc layer was separated andwashed successively with saturated sodium bicarbonate, water and brine.The EtOAc solution was dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by flash chromatography (SiO₂, 50/1 CHCl₃-MeOH to10/1) to afford 1.15 g of the bis-amide (58% yield) as a yellow foam:¹NMR (500 MHz, CDCl₃) δ 8.61 (d, J=8.4 Hz, 2H), 7.29-7.39 (m, 20H), 5.85(bs, 4H), 5.22 (ABq, J=10.0 Hz, Δν=17.3 Hz, 4H), 5.10 (ABq, J=12.2 Hz,Δν=34.3 Hz, 4H), 5.06-5.09 (obs m, 2H), 3.11 (ABq of d, J=17.0, 5.14 Hz,Δν=77.9 Hz, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 170.7 (s), 170.7 (s), 165.4(s), 147.0 (s), 135.7 (s), 135.6 (s), 129.0 (d), 128.9 (d), 128.8 (d),128.75 (d), 128.7 (d), 126.9 (s), 68.0 (t), 67.3 (t), 49.1 (d), 37.0(t). LCMS (50-95% gradient acetonitrile in 0.1% TFA over 10 min), singlepeak retention time=5.97 min on 250 mm column, (M+H)⁺=789.

Step 2

To the product from step 1 (510 mg, 0.65 mmol) was added THF (20 mL) andwater (10 mL). The this stirred mixture was added 10% Pd(C) (500 mg) andammonium formate (1 g). The resulting mixture was heated to 60° C. for 2h and allowed to cool to room temperature. The mixture was filteredthrough celite and concentrated. The resulting material was purifiedagain by reverse phase medium pressure chromatography (C18, 10-70%manual gradient acetonitrile in 0.1% TFA) to afford 137.8 mg (54% yield)of Example 6 as an orange solid: ¹NMR (300 MHz, DMSO-d₆) δ 8.62 (d,J=8.4 Hz, 2H), 6.67 (bs, 4H), 4.725 (dt, J=8.4, 5.4 Hz, 2H), 2.74-2.88(complex m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 172.6 (s), 165.2 (s), 147.0(s), 126.6 (s), 60.8 (t), 49.1 (d). LCMS (5-95% gradient acetonitrile in0.1% TFA over 10 min), single peak retention time=4.01 min on 250 mmcolumn, (M+H)⁺=429. UV/vis (100 μM in PBS) λ_(abs)=433 nm. Fluorescence(100 nM) λ_(ex)=449 nm, λ_(em)=558 nm.

Example 73,6-Diamino-N²,N⁵-bis(14-oxo-2,5,8,11-tetraoxa-15-azaheptadecan-17-yl)pyrazine-2,5-dicarboxamide

To a solution of Example 5 (77.4 mg, 0.15 mmol) in DMF (5 mL) was addedTEA (151 mg, 1.49 mmol) and 2,5-dioxopyrrolidin-1-yl2,5,8,11-tetraoxatetradecan-14-oate (113 mg, 0.34 mmol) and the reactionwas stirred for 16 h at room temperature. The reaction was concentratedand the residue was purified by medium pressure revered phasechromagraphy (LiChroprep RP-18 Lobar (B) 25×310 mm—EMD chemicals 40-63μm, 70 g, 90/10 to 80/20 0.1% TFA-ACN) to afford 37.4 mg (35% yield) ofexample 7 as an orange film: ¹NMR (300 MHz, DMSO-d₆) δ 8.47 (t, J=5.7Hz, 2H), 7.96 (t, J=5.4 Hz, 2H), 3.20-3.60 (complex m, 36H), 3.47 (s,3H), 3.46 (s, 3H), 2.30 (t, J=6.3 Hz, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ170.2 (s), 165.1 (s), 146.0 (s), 126.2 (s), 71.2 (t), 69.7 (t), 69.6(t), 69.5 (t), 69.4 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2 (t). LCMS(5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peakretention time=4.01 min on 250 mm column, (M+H)⁺=719, (M+Na)⁺=741.UV/vis (100 μM in PBS) λ_(abs)=437 nm. Fluorescence (100 nM) λ_(ex)=437nm, λ_(em)=559 nm.

Example 83,6-Diamino-N²,N⁵-bis(26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-yl)pyrazine-2,5-dicarboxamide

To a solution of Example 5 (50.3 mg, 0.10 mmol) in DMF (5 mL) was addedTEA (109 mg, 1.08 mmol) and 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23-octaoxahexacosan-26-oate (128 mg, 0.25 mmol) andthe reaction was stirred for 16 h at room temperature. The reaction wasconcentrated and the residue was purified by medium pressure reveredphase chromagraphy (LiChroprep RP-18 Lobar (B) 25×310 mm—EMD chemicals40-63 μm, ˜70 g, 90/10 to 80/20 0.1% TFA-ACN) to afford 87.9 mg (82%yield) of example 8 as an orange film: ¹NMR (300 MHz, DMSO-d₆) δ 8.46(t, J=5.7 Hz, 2H), 7.96 (t, J=5.4 Hz, 2H), 3.16-3.73 (complex m, 74H),2.28-2.32 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆)—multiple conformations—δ170.1 (s), 169.9 (s) 169.8 (s), 165.1 (s), 146.0 (s), 126.2 (s), 71.2(t), 69.7 (t), 69.6 (t), 69.5 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2(t). LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 min), singlepeak retention time=5.90 min on 250 mm column, (M+H)⁺=1071, (M+2H)⁺=536.UV/vis (100 μM in PBS) λ_(abs)=438 nm. Fluorescence (100 nM) λ_(ex)=438nm, λ_(em)=560 nm.

Example 93,6-Diamino-N²,N⁵-bis(38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azahentetracontan-41-yl)pyrazine-2,5-dicarboxamide

To a solution of Example 5 (53.1 mg, 0.10 mmol) in DMF (5 mL) was addedTEA (114 mg, 1.13 mmol) and 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oate (144mg, 0.21 mmol) in DMF (2.0 mL) and the resulting mixture was stirred for16 h thereafter. The reaction was concentrated and the residue waspurified by medium pressure revered phase chromagraphy (LiChroprep RP-18Lobar (B) 25×310 mm—EMD chemicals 40-63 μm, ˜70 g, 90/10 to 80/20 0.1%TFA-ACN) to afford 87.5 mg (61% yield) of example 9 as an orange film:¹NMR (300 MHz, DMSO-d₆) δ 8.48 (t, J=5.7 Hz, 2H), 7.96 (t, J=5.4 Hz,2H), 7.80-7.86 (m, 2H), 5.94 (bm, 2H), 3.30-3.60 (complex m, 106H),2.26-2.33 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 170.2 (s), 165.1 (s),146.0 (s), 126.2 (s), 71.2 (t), 69.7 (t), 69.6 (t), 69.5 (t), 66.7 (t),58.0 (q), 38.2 (t), 36.2 (t). LCMS (15-95% gradient acetonitrile in 0.1%TFA over 10 min), single peak retention time=5.90 min on 250 mm column,(M+2H)⁺+=712. UV/vis (100 μM in PBS) λ_(abs)=449 nm. Fluorescence (100nM) λ_(ex)=449 nm, λ_(em)=559 nm.

Example 10 Bis(2-(PEG-5000)ethyl)6-(2-(3,6-diamino-5-(2-aminoethylcarbamoyl)pyrazine-2-carboxamido)ethylamino)-6-oxohexane-1,5-diyldicarbamate

A solution of Example 5 (25 mg, 0.049 mmol) in DMF (30 mL) was treatedwith TEA (1 mL) and m-PEG2-NHS (1 g, 0.1 mmol) and the resulting mixturewas stirred for 48 h at room temperature. The mixture was concentratedand the residue was partially purified by gel filtration chromatography(G-25 resin, water). The product was concentrated and further purifiedby reverse phase medium pressure chromatography (C18, 10-70% manualgradient acetonitrile in 0.1% TFA) to afford 137.8 mg (54% yield) ofExample 10 as a tan waxy solid: Maldi MS m/z=11393.

Example 11(R)-2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinoprazine-2-carboxamido)succinicacid

Step 1 Synthesis of 2-amino-5-bromo-3,6-dichloropyrazine

A solution of 2-amino-6-chloropyrazine (25 g, 193.1 mmol) in MeOH (500mL) was treated with NBS (34.3 g, 193.1 mmol), portion-wise, over 1hour. The resulting mixture was stirred for 16 hours thereafter. TLCanalysis at this time shows a small amount of starting materialremaining. Another 1.4 g NBS added and reaction heated to 50° C. for 2hours. The mixture was then cooled to 38° C. and treated with NCS (25.8g, 193.1 mmol). The reaction mixture was heated to 50° C. for 16 hoursthereafter. The mixture was then cooled to room temperature and treatedwith water (500 mL). The precipitate was collected by filtration anddried in a vacuum dessicator to afford 45.4 g (97% yield) of2-amino-5-bromo-3,6-dichloropyrazine as a white solid: ¹³C NMR (75 MHz,CDCl₃) δ 149.9 (s), 145.6 (s), 129.6 (s), 121.5 (s). LCMS (15-95%gradient acetonitrile in 0.1% TFA over 10 min), single peak retentiontime=4.51 min on 30 mm column, (M+H)⁺=244, (M+H+ACN)⁺=285.

Step 2 Synthesis of 5-amino-3,6-dichloropyrazine-2-carbonitrile

A mixture of CuCN (8.62 g, 96.3 mmol) and NaCN (4.72 g, 96.3 mmol) washeated under high vacuum to 90° C. The resulting mixture was subjectedto three Argon/Vacuum cycles and placed under a final positive pressureof Argon. The mixture was allowed to cool to room temperature and DMF(150 mL) was added. The heterogenous mixture was heated to 130° C. for2.5 hours. To the resulting homogeneous mixture of sodium dicyanocupratewas added a solution of the product from step 1 (15.6 g, 64.2 mmol)dissolved in DMF (150 mL), dropwise, over 1 hour. The temperature wasgradually raised to 150° C. and the resulting mixture was stirred atthis temperature for 10 hours thereafter. The reaction was then allowedto cool to room temperature and poured into water (1 L). The resultingmixture was extracted with EtOAc (3×) and the combined extracts werefiltered to remove a flocculant dark solid, washed with brine, dried(Na₂SO₄), filtered again and concentrated. Purification by flash columnchromatography (SiO₂, 10/1 hexanes-EtOAc to 3/1) to afford 6.70 g (55%yield) of the nitrile product as a tan solid: ¹³C NMR (75 MHz, CDCl₃) δ153.9 (s), 149.1 (s), 131.7 (s), 115.4 (s), 111.0 (s). GCMS (inj.temperature=280° C., 1.0 mL/min helium flow rate, temperature program:100° C. (2 min hold), ramp to 300° C. 10° C./min (2 min hold), majorpeak retention time=16.556 min, m/z (EI)=188, 190.

Step 3 Synthesis of5-amino-3-(bis(2-methoxyethyl)amino)-6-chloropyrazine-2-carbonitrile

To the product from step 2 (1.00 g, 5.29 mmol) in ACN (20 mL) was addedbis(2-methoxyethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and the reactionmixture was heated to 70° C. for 16 hours thereafter. The reaction wascooled and concentrated. The residue was partitioned with EtOAc andwater. The organic layer was separated and the aqueous was extractedagain with EtOAc. The combined organic extracts were washed with brine,dried (Na₂SO₄), filtered and concentrated. Purification by flash columnchromatography (SiO₂, 10/1 hexanes-EtOAc to 1/1) afforded 950 mg (63%yield) of the desired adduct as a yellow solid: ¹NMR (300 MHz, CDCl₃) δ7.47 (bs, 2H), 3.77 (t, J=5.7 Hz, 4H), 3.52 (t, J=5.4 Hz, 4H), 3.25 (s,6H). ¹³C NMR (75 MHz, CDCl₃) δ 154.7 (s), 152.0 (s), 120.9 (s), 119.5(s), 95.8 (s), 71.0 (t), 59.1 (q), 50.0 (t). LCMS (50-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=4.91min on 250 mm column, (M+H)⁺=286, (M+Na)⁺=308, (M+Na+ACN)⁺=349.

Step 4 Synthesis of3-(bis(2-methoxyethyl)amino)-5-bromo-6-chloropyrazine-2-carbonitrile

To the product from step 3 (1.39 g, 4.88 mmol) in 48% hydrobromic acid(20 mL) at 0° C. (ice-salt bath), was added a solution of sodium nitrite(673 mg, 9.75 mmol) in water (10 mL) dropwise over 30 min. The resultingmixture was stirred at 0-5° C. for 1 h and poured into a stirredsolution of CuBr₂ (1.64 g, 7.34 mmol) in water (100 mL). The resultingmixture was stirred for 16 h at room temperature thereafter. The mixturewas extracted with EtOAc (3×). The combined organic layers were dried(Na₂SO₄), filtered and concentrated. Purification by flash columnchromatography (SiO₂, 50/1 CHCl₃-MeOH) afforded 1.00 g (58% yield) ofthe bromide as a orange-brown solid: ¹NMR (300 MHz, CDCl₃) δ 3.99 (t,J=5.4 Hz, 4H), 3.64 (t, J=5.4 Hz, 4H), 3.35 (s, 6H). ¹³C NMR (75 MHz,CDCl₃) δ 152.8 (s), 140.8 (s), 133.4 (s), 117.2 (s), 108.3 (s), 70.4(t), 59.1 (t), 50.5 (q). LCMS (50-95% gradient acetonitrile in 0.1% TFAover 10 min), single peak retention time=4.55 min on 250 mm column,(M+H)⁺=349, 351.

Step 5 Synthesis of3-(bis(2-methoxethyl)amino)-6-chloro-5-(furan-2-yl)pyrazine-2-carbonitrile

A mixture of the product from step 4 (1.0 g, 2.87 mmol), 2-furanboronicacid (643 mg, 5.75 mmol), Cs₂CO₃ (3.31 g, 10.2 mmol), TFP (35 mol %, 236mg, 1.02 mmol), and Pd₂ dba₃-CHCl₃ (5 mol %, 10 mol % Pd, 150 mg) wassubjected to 3 vacuum/Argon cycles and placed under a positive pressureof Argon. Anhydrous dioxane (50 mL) was added and the reaction mixturewas heated to 75° C. for 16 h thereafter. The reaction mixture wascooled to room temperature, diluted with EtOAc (100 mL) and filteredthrough a medium frit. Concentration and purification of the residue byflash chromatography (SiO₂, 50/1 CHCl₃-MeOH) afforded the 757 mg of thefuran adduct (78% yield) as a tan powder: LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=6.41min on 250 mm column, (M+H)⁺=337.

Step 6 Synthesis of6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxylic acid

To a well stirred mixture of ACN (11 mL), CCl₄ (7 mL), and water (11 mL)were added sodium periodate (1.07 g, 5.00 mmol) and RuO2.H₂O (13.3 mg,0.10 mmol), sequentially. The resulting mixture was stirred vigorouslyat room temperature for 30 min and treated with sodium bicarbonate (2.10g, 25.0 mmol) followed by water (5 mL). Vigorous stirring for another 15minutes was followed by the addition of a solution of the product fromStep 5 (276 mg, 0.82 mmol) dissolved in ACN (1 mL). The green mixturewas stirred at room temperature for 5.5 h. The mixture was transferredto a separatory funnel and extracted with EtOAc. The aqueous layer wasadjusted to pH˜3.5 and extracted again with EtOAc (2×). The combinedextracts were washed with 20% sodium bisulfite and brine and dried(Na₂SO₄). Filtration and concentration afforded 140 mg (54% yield) ofcarboxylic acid as a pale yellow solid: LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=5.05min on 250 mm column, (M+H)⁺=315.

Step 7 Synthesis of (R)-dibenzyl2-(6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxamido)succinate

A mixture of the product from step 6 (140 mg, 0.45 mmol), EDC-HCl (128mg, 0.67 mmol) and HOBt.H₂O (102 mg, 0.67 mmol) in anhydrous DMF (25 mL)was stirred together at room temperature for 30 min. To this stirredmixture was added (R)-dibenzyl 2-aminosuccinate p-TsOH salt (213 mg,0.44 mmol) followed by TEA (1 mL). The resulting mixture was stirred for16 h thereafter. The reaction mixture was concentrated and partitionedwith EtOAc and saturated sodium bicarbonate solution. The EtOAc layerwas separated and washed with saturated sodium bicarbonate and brine,dried (Na₂SO₄), filtered and concentrated to afford 240 mg (88% yield)of the pyrazine amide as an orange foam: LCMS (15-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=8.76min on 250 mm column, (M+H)=610, (M+Na)⁺=632.

Step 8 (R)-dibenzyl2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamido)succinate

To the product from step 7 (240 mg, 0.39 mmol) was added morpholine (5mL). The reaction mixture was heated to 70° C. for 2 h. The mixture wascooled and concentrated. The residue was partitioned with EtOAc andwater. The EtOAc layer was separated and washed with saturated sodiumbicarbonate and brine. The EtOAc layer was dried (Na₂SO₄), filtered andconcentrated. Purification by flash column chromatography (SiO₂, 3:1 to1:1 hexanes-EtOAc) afforded 199 mg (75% yield) of the morpholine adductas an orange foam: LCMS (15-95% gradient acetonitrile in 0.1% TFA over10 min), single peak retention time=8.76 min on 250 mm column,(M+H)⁺=661, (M+Na)⁺=683.

Step 9 Synthesis of Example 11

The dibenzyl ester (115 mg, 0.17 mmol) in THF (10 mL) was added 1.0Nsodium hydroxide (4 mL). The mixture was stirred for 1 h at roomtemperature. The pH was adjusted to ˜2 with 1.0N HCl and the solutionwas concentrated. Purification of the residue by medium pressurereversed phase chromatography (LiChroprep RP-18 Lobar (B) 25×310 mm—EMDchemicals 40-63 μm, ˜70 g, 90/10 to 50/50 0.1% TFA-ACN) afforded 32 mg(27% yield) of example 11 as an orange solid: LCMS (15-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=4.47min on 250 mm column, (M+H)⁺=481. UV/vis (100 μM in PBS) λ_(abs)=438 nm.Fluorescence (100 nM) λ_(ex)=449 nm, λ_(em)=⁵⁷⁰ nm.

Example 12 Protocol for Assessing Renal Function

An example of an in vivo renal monitoring assembly 10 is shown in FIG. 2and includes a light source 12 and a data processing system 14. Thelight source 12 generally includes or is interconnected with anappropriate device for exposing at least a portion of a patient's bodyto light therefrom. Examples of appropriate devices that may beinterconnected with or be a part of the light source 12 include, but arenot limited to, catheters, endoscopes, fiber optics, ear clips, handbands, head bands, forehead sensors, surface coils, and finger probes.Indeed, any of a number of devices capable of emitting visible and/ornear infrared light of the light source may be employed in the renalmonitoring assembly 10.

Still referring to FIG. 2, the data processing system 14 of the renalmonitoring assembly may be any appropriate system capable of detectingspectral energy and processing data indicative of the spectral energy.For instance, the data processing system 14 may include one or morelenses (e.g., to direct and/or focus spectral energy), one or morefilters (e.g., to fitter out undesired wavelengths of spectral energy),a photodiode (e.g., to collect the spectral energy and convert the sameinto electrical signal indicative of the detected spectral energy), anamplifier (e.g., to amplify electrical signal from the photodiode), anda processing unit (e.g., to process the electrical signal from thephotodiode). This data processing system 14 is preferably configured tomanipulate collected spectral data and generate an intensity/timeprofile and/or a concentration/time curve indicative of renal clearanceof a pyrazine derivative of the present invention from the patient 20.Indeed, the data processing system 14 may be configured to generateappropriate renal function data by comparing differences in manners inwhich normal and impaired cells remove the pyrazine derivative from thebloodstream, to determine a rate or an accumulation of the pyrazinederivative in organs or tissues of the patient 20, and/or to providetomographic images of organs or tissues having the pyrazine derivativeassociated therewith.

In one protocol for determining renal function, an effective amount of apyrazine derivative of the invention is administered to the patient(e.g., in the form for a pharmaceutically acceptable composition). Atleast a portion of the body of the patient 20 is exposed to visibleand/or near infrared light from the light source 12 as indicated byarrow 16. For instance, the light from the light source 12 may bedelivered via a fiber optic that is affixed to an ear of the patient 20.The patient may be exposed to the light from the light source 12 beforeor after administration of the pyrazine derivative to the patient 20. Insome cases, it may be beneficial to generate a background or baselinereading of light being emitted from the body of the patient 20 (due toexposure to the light from the light source 12) before administering thepyrazine derivative to the patient 20. When the pyrazine derivative thatis in the body of the patient 20 is exposed to the light from the lightsource 12, the pyrazine derivative emanates light (indicated by arrow18) that is detected/collected by the data processing system 14.Initially, administration of the pyrazine derivative to the patient 20generally enables an initial spectral signal indicative of the initialcontent of the pyrazine derivative in the patient 20. The spectralsignal then tends to decay as a function of time as the pyrazinederivative is cleared from the patient 20. This decay in the spectralsignal as a function of time is indicative of the patient's renalfunction. For example, in a first patient exhibiting healthy/normalrenal function, the spectral signal may decay back to a baseline in atime of T. However, a spectral signal indicative of a second patientexhibiting deficient renal function may decay back to a baseline in atime of T+4 hours. As such, the patient 20 may be exposed to the lightfrom the light source 12 for any amount of time appropriate forproviding the desired renal function data. Likewise, the data processingsystem 14 may be allowed to collect/detect spectral energy for anyamount of time appropriate for providing the desired renal functiondata.

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What is claimed is:
 1. A method of using a compound, the methodcomprising: administering the compound into a body of a patient;exposing the compound that is in the body of the patient to visible orinfrared red light, thereby causing spectral energy to emanate from thecompound; detecting the spectral energy emanated from the compound inthe body; and assessing renal function of the patient based on thedetected spectral energy, the compound being of Formula I, wherein:

each of X¹ and X² is independently —CN, —CO₂R¹, —CONR²R³, or —CONH(PS);each of Y¹ and Y² is independently —NR⁴R⁵ or

Z¹ is a single bond, —CR¹⁸R¹⁹—, —O—, —NR²⁰—, —NCOR²¹—, —S—, —SO—, or—SO₂—; each of R¹ to R⁵ and R¹⁸ to R²¹ is independently —H,—CH₂(CHOH)_(a)R⁴⁴, —CH₂(CHOH)_(a)CO₂H, —(CHCO₂H)_(a)CO₂H,—(CH₂CH₂O)_(c)R⁴⁹, —(CH₂)_(a)SO₃H, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)NHSO₃H,—(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻, or —(CH₂)_(a)PO₃ ⁼;each of R⁴⁴ and R⁴⁹ is independently —H or —CH₃; (PS) is a sulfated ornon-sulfated polysaccharide chain comprising one or more monosaccharideunits connected by glycosidic linkages; and ‘a’ is an integer from 1 to10, ‘c’ is an integer from 1 to 100, and each of ‘m’ and ‘n’independently is an integer from 1 to
 3. 2. The method of claim 1,wherein said spectral energy is fluorescence from said compoundcomprising visible or infrared light.
 3. The method of claim 1, whereinsaid step of assessing renal function of the patient based on thedetected spectral energy comprises using data indicative of the detectedspectral energy and generating an intensity/time profile indicative ofclearance of the compound from the body of the patient.
 4. The method ofclaim 1, wherein said step of assessing renal function of the patientbased on the detected spectral energy comprises: comparing differencesin which normal and impaired cells of the patient remove the compoundfrom the bloodstream; measuring a rate or an accumulation of thecompound in an organ or a tissue of the patient; or obtaining atomographic image of an organ or a tissue of the patient having thecompound associated therewith.
 5. The method of claim 1, wherein each ofX¹ and X² are —CONR²R³, and each of Y¹ and Y² is —NR⁴R⁵.
 6. The methodof claim 5, wherein each of R² and R⁴ is —H, and R³ is —CH₂(CHOH)_(a)R⁴⁴or —(CH₂CH₂O)_(c)R⁴⁹, and R⁵ is —H or —(CH₂CH₂O)_(c)R⁴⁹.
 7. The methodof claim 6, wherein R³ is —(CH₂CH₂O)_(c)R⁴⁹, and R⁵ is —H.
 8. The methodof claim 7, wherein R⁴⁹ is —CH₃, and ‘c’ is an integer from 1 to
 20. 9.The method of claim 1, wherein each of X¹ and X² is —CO₂R¹, and each ofY¹ and Y² is —NR⁴R⁵.
 10. The method of claim 9, wherein each of R¹ andR⁴ is —H, and R⁵ is —CH₂(CHOH)_(a)R⁴⁴ or —(CH₂CH₂O)_(c)R⁴⁹.
 11. Themethod of claim 10, wherein R⁴⁴ is —H, R⁴⁹ is —CH₃, ‘a’ is an integerfrom 1 to 6, and ‘c’ is an integer from 1 to
 20. 12. The method of claim1, wherein each of X¹ and X² is —CN, and each of Y¹ and Y² is —NR⁴R⁵.13. The method of claim 12, wherein R⁴ is —H, and R⁵ is—CH₂(CHOH)_(a)R⁴⁴ or —(CH₂CH₂O)_(c)R⁴⁹.
 14. The method of claim 13,wherein R⁴⁴ is —H, R⁴⁹ is —CH₃, ‘a’ is an integer from 1 to 6, and ‘c’is an integer from 1 to
 20. 15. The method of claim 1, each of X¹ and X²is —CONH(PS), and each of Y¹ and Y² is —NR⁴R⁵.
 16. The method of claim15, wherein each of R⁴ and R⁵ is independently —H or —CH₂(CHOH)_(a)R⁴⁴.17. The method of claim 16, wherein each of R⁴, and R⁵ is —H.
 18. Themethod of claim 15, wherein the one or more monosaccharide units of thepolysaccharide chain (PS) are selected from the group consisting ofglucose, fructose, mannose, xylose, and ribose.